Functional transcriptomics of a migrating cell in Caenorhabditis elegans

Abstract

In both metazoan development and metastatic cancer, migrating cells must carry out a detailed, complex program of sensing cues, binding substrates, and moving their cytoskeletons. The linker cell in Caenorhabditis elegans males undergoes a stereotyped migration that guides gonad organogenesis, occurs with precise timing, and requires the nuclear hormone receptor NHR-67. To better understand how this occurs, we performed RNA-seq of individually staged and dissected linker cells, comparing transcriptomes from linker cells of third-stage (L3) larvae, fourth-stage (L4) larvae, and nhr-67-RNAi-treated L4 larvae. We observed expression of 8,000-10,000 genes in the linker cell, 22-25% of which were up- or down-regulated 20-fold during development by NHR-67. Of genes that we tested by RNAi, 22% (45 of 204) were required for normal shape and migration, suggesting that many NHR-67-dependent, linker cell-enriched genes play roles in this migration. One unexpected class of genes up-regulated by NHR-67 was tandem pore potassium channels, which are required for normal linker-cell migration. We also found phenotypes for genes with human orthologs but no previously described migratory function. Our results provide an extensive catalog of genes that act in a migrating cell, identify unique molecular functions involved in nematode cell migration, and suggest similar functions in humans.

Fig. 1.

LC biology, dissection, and transcriptional profiling. (A) The LC position and gonad shape during LC migration. The LC (red), located at the tip of the gonad (blue), begins migrating toward the head, performs a U-turn while switching to the dorsal bodywall, migrates posteriorly and turns downward, and continues to migrate toward the tail until stopping at the cloaca (blue line). The bottom panel shows LC migration in an nhr-67(RNAi) animal at the same L4 stage timepoint as the wild-type animal. Stars indicate the types of LCs collected for profiling. (B) A YFP-labeled LC is dissected from an animal glued to an agar pad by extruding the gonad with a cutting needle and collecting the LC into a patch pipet. (Scale bar, 20 μm.) (C) Three classes of LCs microdissected for RT-PCR and RNA-seq. Trailing gonads are outlined in yellow. The nhr-67(RNAi) L4-stage LC is morphologically retarded, still showing the spheroid shape normally seen in L3-stage LCs. (Scale bar, 20 μm.) (D) The RNA-seq profile for nhr-67 showed tissue-specificity of transcripts and efficacy of RNAi. Our RT-PCR protocol (13) deliberately limited amplification to the 3′ exons of genes.

Fig. 2.

Overall gene-expression patterns in LCs. (A) k-partitioning of the expression data from LC genes. Expression data from 8,011 genes expressed in pooled wild-type LCs (“LC genes”) were compared between LC types [L3, L4, nhr-67(RNAi) L4], and mixed-stage larvae (SI Appendix, Table S1 and Dataset S1). This process distinguished four sets of variably expressed genes from a fifth set of 1,164 ubiquitously expressed genes. Ubiquitous genes disproportionately encoded such housekeeping functions as protein translation and electron transport (SI Appendix, Table S7). Yellow and blue denote high and low expression levels, respectively; ubiquitous genes are labeled in red. Genes belonging to each partition are listed in Dataset S1. An alternative partitioning is shown in SI Appendix, Fig. S1. (B) LC/larval expression ratio versus maximum LC gene expression for LC genes. A set of 1,097 LC-enriched genes, including nhr-67, showed ≥20× more expression in wild-type LCs than in larvae, and are marked in blue. Two genes previously shown to be active in the LC (3), the netrin receptor unc-5 and the metalloprotease zmp-1, show more moderate LC-enrichment; conversely, the cohesin subunit gene him-1 (the activity of which in LCs we confirmed with both antibody staining and RNAi; SI Appendix, Table S9) is an instance of the LC-enriched class. Ubiquitous genes from A are shown in red; other, unenriched LC genes are in gray. (C) L3- vs. L4-stage expression for LC genes. Ubiquitous genes are easily distinguished from LC-enriched genes, which are more likely to show moderate and stage-specific expression. MSP and MSD genes (green and yellow) are strongly up-regulated at the L4 stage. (D) A comparison, for LC genes, of the ratio of L4-stage to L3-stage gene expression to the ratio of L4-stage to nhr-67(RNAi) L4-stage expression. Of LC-expressed genes, 25% showed ≥20-fold expression changes for which development and NHR-67 activity were correlated (SI Appendix, Table S3). Subsets of LC genes encoding potassium channels, protein kinases, and regulators of muscle contraction are shown. All three subsets are visibly overrepresented among genes up-regulated by NHR-67 in L4-stage LCs. The genes along the ascending diagonal have significant expression in wild-type L4-stage LCs, but no detectable expression in either wild-type L3-stage LCs or nhr-67(RNAi) L4-stage LCs; genes with no expression were given a nominal expression level of 0.01 RPKM.

Fig. 3.

Types of LC migration phenotypes induced by RNAi. (Scale bar, 20 μm: applies to all panels except abnormal shapes panel, which is 10 μm.)

Fig. 4.

srsx-18, a seven-transmembrane receptor, is a likely effector of NHR-67. (A) The LC migrates normally until near the end of the migration (early L4 stage, Upper two panels), but then slows down in srsx-18(RNAi) males. By the time the wild-type males have completed migration in the mid-L4 stage, srsx-18(RNAi) males are still migrating (Lower two panels). Note that delayed migration is one of nhr-67’s RNAi phenotypes (3). Arrow, LC; parallel lines, outline of gonad; single line, cloaca. (B) RNA-seq shows srsx-18 is transcriptionally active solely in wild-type L4-stage LCs, not in L3-stage LCs or in nhr-67(RNAi) L4-stage LCs. This L4-stage expression is regulated by nhr-67. (C and D) srsx-18::YFP is absent in the L3-stage but expressed in the L4-stage LC. (E) srsx-18::YFP expression in the L4-stage LC is abolished by nhr-67(RNAi). Other gene expression reporters are described in SI Appendix, Table S10.

Fig. 5.

An overview of recently identified genes functioning in the LC, with possible functions in migration. Up- and down-regulation of genes during LC development by NHR-67 is shown, along with many genes up-regulated by NHR-67 and discussed in the main text. Genes marked in blue have ≥20-fold higher expression in L4-stage than in L3-stage LCs; genes marked in red are higher in L3-stage than L4; genes in black have a <20-fold difference. Further LC gene functions are given in SI Appendix, Tables S11 and S12.